Extracellular Connective Tissue Research Articles

The metathoracic wing-hinge chordotonal organ of an atympanate moth, Actias luna (Lepidoptera, Saturniidae): a light- and electron-microscopic study.

The structure of a simple chordotonal organ, the presumed homologue of the noctuoid moth tympanal organ, is described in the atympanate moth, Actias luna. The organ consists of a proximal scolopidial region and a distal strand, which attaches peripherally to the membraneous cuticle ventral to the hindwing alula. The strand is composed of elongate, microtubule-rich cells encased in an extracellular connective tissue sheath. The scolopidial region houses three mononematic, monodynal scolopidia, each comprised of a sensory cell, scolopale cell, and attachment cell. The dendritic apex is octagonally shaped in transverse section, its inner membrane lined by a laminated structure reminiscent of the noctuoid tympanal organ 'collar'. A 9 + 0-type cilium emerges from the dendritic apex, passes through both the scolopale lumen and cap, and terminates in an extracellular space distal to the latter. Proximal extensions of the attachment cell and distal prolongations of the scolopale cell surrounding the cap are joined by an elaborate desmosome, with which is associated an extensive electron-dense fibrillar plaque. Within the scolopale cell, this plaque constitutes the scolopale 'rod' material. The data are discussed in terms of both the organ's potential function, and its significance as the evolutionary prototype of the noctuoid moth ear.

Computer assisted quantitation of terminal hepatic vein connective tissue in the rat

Computer assisted image analysis has been used to quantify the cellular and extracellular connective tissue component of rat liver terminal hepatic venules, in control animals and those exposed to 40% ethanol in drinking water. A significant relationship existed between the size of the terminal hepatic venule and the amount of connective tissue it contained in 14 of 15 controls and 17 of 18 ethanol exposed rats. Thickening of the terminal hepatic vein wall assessed to be present in ethanol treated rats by direct observation was confirmed by image analysis in all cases (p < 0.01). Significant differences between treated and control livers (p < 0.05) were detected by image analysis when not apparent to human observers. Sensitive quantitative assessment of terminal hepatic vein wall thickening was thus achieved by computerized analysis of liver sections.

Corrélation entre la maladie de Dupuytren et l'arc cornéen : la dyslipidémie peut-elle être un facteur étiopathogénique commun ?

Because of the frequent presence of corneal arcus senilis in patients affected by Dupuytren's disease in order to evaluate this association, the authors conducted a biomicroscopic examination of the cornea in 336 patients treated surgically for Dupuytren's disease, at the Hand Surgery Unit of the University of Modena from November 1985 to December 1989. They observed corneal arcus senilis in 259 patients, i.e. in 77.1% of patients with Dupuytren's disease. Due to the statistically significant correlation between arcus senilis and hyperlipidemia as reported by Tschetter (1980) and Felder (1981), the Authors collected a blood sample from all 336 patients to evaluate serum cholesterol and tryglicerides. This study revealed a dyslipidemia in 54.8% of patients with Dupuytren's disease and in 60.2% of patients suffering from both Dupuytren's disease and arcus senilis. Because of the high frequency of dislipidemia in patients with Dupuytren's disease and arcus senilis, which are apparently two well-distinguished disease, the authors suggest that a lipid disorder may be a common aetiopathogenic factor. In particular, in favour of the possible role of hyperlipidemia in Dupuytren's disease, Electron Microscope Studies revealed lipid inclusions within fibroblasts and in the extracellular connective tissue of all pathologic palmar aponeurosis from 11 patients with Dupuytren's disease: these lipid inclusions were never seen in the normal aponeurosis taken from 5 control patients treated for traumatic palmar injuries.

Dietary and mechanically induced rabbit Iliac-femoral atherosclerotic lesions: A chemical and morphologic evaluation

The effect of combining chronic endothelial injury and intermittent meal feeding of a high and low cholesterol, coconut oil, peanut oil diet on plasma lipid and lipoprotein content and on the formation of atherosclerotic lesions within the iliac-femoral artery of rabbits was studied. Alternate feeding of a 1 or 0.1% cholesterol, 3% coconut oil, 3% peanut oil diet for 3 to 14 weeks resulted in a 4- to 11-fold increase in plasma cholesterol with 59 to 79% of the plasma cholesterol eluting in a molecular weight fraction comparable to human low density lipoproteins (LDL). In the iliac-femoral artery, an atherosclerotic intimal lesion with an average cross-sectional area of 0.452 mm 2 was present in 98% of the animals. The lesion was typically eccentric in location and contained both superficial- and deep-intimal lipid-filled monocyte-macrophages, extracellular lipid, smooth muscle cells, and extracellular connective tissue matrix. The relative percent lipid composition of the iliac-femoral lesion was 62% cholesteryl ester, 21% free cholesterol, and 17% phospholipid. Thus, we conclude that the combination of meal feeding a cholesterol/fat diet, dietary regimen and chronic mild endothelial injury in the rabbit results in (1) a diet-induced hypercholesterolemia in which LDL appear to be the predominant lipoprotein; and (2) a lesion within the iliac-femoral artery comparable in histologic and chemical composition to a human fatty streak.

A novel polyclonal antibody (CL-B1/29) for immunolocalization of transforming growth factor-beta 2 (TGF-beta 2) in adult mouse.

A polyclonal antibody (CL-B1/29) raised against a synthetic peptide with an amino acid sequence identical to the first 29 N-terminal residues of bovine bone-derived transforming growth factor-beta 2 (TGF-beta 2) was characterized and used for immunolocalization of TGF-beta 2 in adult mice. Reduced staining of immunoblots and tissue after absorption of the antiserum with the immunizing peptide or with TGF-beta 2 but not with purified TGF-beta 1 demonstrated that the reagent is specific for TGF-beta 2, with little or no crossreactivity with TGF-beta 1. The immunolocalization of TGF-beta 2 was investigated in formalin-fixed, paraffin-embedded cultured cells and murine tissue. Specimens pre-digested with testicular hyaluronidase demonstrated immunostaining predominantly of extracellular connective tissue matrix, whereas specimens pre-digested with pronase E demonstrated primarily cytoplasmic staining. Immunoreactivity was widely distributed in connective tissue, muscle, adsorptive and secretory epithelia, especially of endocrine tissue, and neural tissue of adult mice.

Spatial arrangements of microfibrils in myocardial scars: Application of antibody to fibrillin

Acute myocardial infarction kills myocytes; viable and necrotic myocytes disconnect and the ends of the viable cells become anchored to collagen fibers during reparative scar tissue formation. These anchorages have not been examined in detail, although previous studies have shown that microfibrils (MFs) concentrate at the edges of scars and at the tips of normal papillary muscles. We examined the spatial arrangements of MFs at these two sites in human hearts. Light and electron microscopic observations were made on tissue samples oriented in the long axis of myocytes and stained with monoclonal antibodies to fibrillin, a glycoprotein component of human microfibrils. MFs had identical arrangements at both sites, where they formed fibrous connections between myofibers and collagen fibers. These connections were oriented in the long axis of the muscle cells. At the myocyte ends, MFs appeared to intertwine with MFs in the normal endomysium; in the main body of the connections, MFs formed compact, 200 to 500 nm thick, fibrillin-positive fibers; and at the collagen ends, MFs splayed out among collagen fibrils. These observations indicate that MFs form myofiber-collagen fiber linkages at sites where the power of myocardial contraction is being transmitted to the extracellular connective tissue framework. Formation of such linkages seems to be an important step in the successful repair of necrotic myocardial lesions.

Cytoarchitecture of the smooth muscles and pericytes of rat cerebral blood vessels

The three-dimensional cytoarchitecture of the smooth muscles and pericytes of rat cerebral blood vessels was studied by scanning electron microscopy after removing extracellular connective tissue matrices with the KOH-collagenase digestion method. The tunica media of major intracranial arteries such as the internal carotid, vertebral, basilar, and other cerebral arteries measuring more than 100 microns in outer diameter consisted of spindle-shaped smooth-muscle cells arranged circularly to the long axis of the vessel. Muscle cells at the branching points, however, showed a variety of shapes, sizes, and arrangements. As the vessel size decreased, smooth-muscle cells showed bi- or trifurcations at the cell poles. In the precapillary arterioles, smooth-muscle cells which had helically surrounded the endothelial tubes had bulging cell bodies with various cytoplasmic processes extending from the cell poles. Distinct specializations presumed to be sphincters were not found on the arteries or arterioles. Pericytes of the capillary had become extended along the vessel axis, having fusiform cell bodies with longitudinally oriented long cytoplasmic processes. Cells located periendothelially in the venules and veins were stellate in shape with many cytoplasmic processes which were interwoven to form complicated cellular networks around the endothelial tube.

The three-dimensional organization and ultrastructure of lymphatics in the rat intestinal mucosa as revealed by scanning electron microscopy after KOH-collagenase treatment.

The three-dimensional organization and ultrastructure of lymphatics in the rat intestinal mucosa were studied by scanning electron microscopy using the KOH-collagenase digestion method (to remove extracellular connective tissue matrices) and by conventional transmission electron microscopy. Each villus possessed three or more lymphatics in the deeper layer of the lamina propria. These initial lymphatics were 5-15 microns in diameter and ran parallel to each other in the longitudinal direction of the villus. These lymphatics were free from adhering periendothelial cells, but had numerous cytoplasmic folds and projections of the endothelium on their abluminal surface. At the villous base, the lymphatics emptied into several thicker (about 70-80 microns) lymphatics, which further connected together to form a flat and wide sinus (intra-villous lymphatic sinus). This sinus was relatively smooth in abluminal surface. From the bottom of each sinus, several lymphatics descended perpendicularly to drain into lymphatics in the submucosa. The villous lymphatics described above seemed to be partially accompanied by bundles of smooth muscle cells, which ran longitudinally in the villous core. The submucosal lymphatics were smooth in abluminal surface and possessed no periendothelial cells. These lymphatics ran in a transverse direction just beneath the muscularis mucosae to form a two-dimensional mesh-work by anastomosing with one another. This submucosal lymphatic network was often closely associated with the muscle cells of the muscularis mucosae, but was not accompanied by submucosal arteries.

Extracellular matrix of the skin: 50 years of progress.

The extracellular connective tissue matrix of the skin is a complex aggregate of distinct collagenous and non-collagenous components. Optimal quantities and delicate interactions of these components are necessary to maintain normal physiologic properties of skin. This overview summarizes the progress made in understanding the normal biology and biochemistry of the extracellular matrix, and will highlight cutaneous diseases with underlying molecular defects in the structure and expression of extracellular matrix components.

Scanning electron microscopy of the muscle coat of the guinea-pig small intestine.

We have studied the layers of the muscular coat of the guinea-pig small intestine after enzymatic and chemical removal of extracellular connective tissue. The cells of the longitudinal muscle layer are wider, have rougher surfaces, more finger-like processes and more complex terminations, but fewer intercellular junctions than cells in the circular muscle layer. A special layer of wide, flat cells with a dense innervation exists at the inner margin of the circular muscle layer, facing the submucosa. The ganglia of the myenteric and submucosal plexuses are covered by a smooth basal lamina, a delicate feltwork of collagen fibrils, and innumerable connective tissue cells. The neuronal and glial cell processes at the surface of ganglia form an interlocking mosaic, which is loosely packed in newborn and young animals, but becomes tightly packed in adults. The arrangement of glial cells becomes progressively looser along finer nerve bundles. Single varicose nerve fibres are rarely exposed, but multiaxonal bundles are common. Fibroblast-like cells of characteristic shape and orientation are found in the serosa; around nerve ganglia; in the intermuscular connective tissue layer and in the circular muscle, where they bridge nerve bundles and muscle cells; at the submucosal face of the special, flattened inner circular muscle layer; and in the submucosa. Some of these fibroblast-like cells correspond to interstitial cells of Cajal. Other structures readily visualized by scanning electron microscopy are blood and lymphatic vessels and their periendothelial cells. The relationship of cellular elements to connective tissue was studied with three different preparative procedures: (1) freeze-cracked specimens of intact, undigested intestine; (2) 'stretch preparations' of longitudinal muscle with adhering myenteric plexus; (3) sheets of submucosal collagen bundles from which all cellular elements had been removed by prolonged detergent extraction.

NEUTROPHILS FROM SUBJECTS WITH CHRONIC OBSTRUCTIVE LUNG DISEASE SHOW ENHANCED CHEMOTAXIS AND EXTRACELLULAR PROTEOLYSIS

Peripheral polymorphonuclear leucocytes (PMN) from subjects with emphysema or bronchiectasis digested significantly more iodine-125-labelled fibronectin (on average, 250% and 280%, respectively) than did those from control subjects. PMN from patients with bronchiectasis contained significantly more of the serine proteinase elastase than did the control cells, which may have contributed to their greater extracellular proteolysis. PMN from patients with emphysema, but not those with bronchiectasis, showed enhanced chemotaxis (on average 260%) in response to a chemotactic peptide compared with control cells. Thus, PMN from subjects with chronic obstructive lung diseases can digest more extracellular connective tissue protein than PMN from healthy subjects. This behaviour suggests a mechanism for the pathological tissue damage associated with these disorders. Furthermore, the sensitivity to chemotactic factors of PMN from emphysematous patients would contribute to the larger numbers of these cells in their lung tissues, thus increasing further the proteolytic burden in the lungs.

Association of heparan sulfate proteoglycan and laminin with the cytoskeleton in rat liver.

Rats were injected with 35SO4 and after 2 h their livers were removed and used to prepare a detergent-insoluble cytoskeleton fraction. Spectrin, cytokeratins, and actin were major protein components of the isolated cytoskeletons. The cytoskeleton fraction accounted for approximately 14% of the total trichloroacetic acid-insoluble 35SO4 radioactivity incorporated into the liver. The cytoskeleton-associated radioactivity was present in a single species of macromolecule. This molecule was not present to a significant extent in the detergent-soluble fraction containing the cell supernatant and dissolved membrane proteins. Further characterization revealed the cytoskeleton-associated molecule was a heparan sulfate proteoglycan: it was eluted from a Sepharose CL-4B column under denaturing conditions at Kav = 0.4; following mild alkaline hydrolysis the radioactivity was eluted at a Kav = 0.7; when this material was subjected to nitrous acid hydrolysis all of the radioactivity was eluted near the column included volume. The isolated cytoskeletons contained attached nuclei. Pure nuclei isolated without associated cytoskeletal elements contained less than 1% of the total liver trichloroacetic acid-insoluble 35SO4 radioactivity and no detectable heparan sulfate proteoglycan. These results suggested that other matrix proteins might be associated with the liver cytoskeleton. When the subcellular distribution of laminin was monitored by immunostaining proteins transferred to nitrocellulose, laminin was detected exclusively in the cytoskeleton fraction. These results provide evidence for an association between extracellular connective tissue proteins and intracellular structural proteins.

An ultrastructural study of the response of hamster bronchial epithelium to human neutrophil elastase.

The central intrapulmonary bronchi of hamsters were examined by transmission electron microscopy at varying times following intratracheal instillation of human neutrophil elastase (HNE) or its vehicle, saline. Two hours after HNE treatment, there was a marked irregularity of the surfaces of many nonciliated epithelial cells; a differential count of transepithelial cells (those with both a basal lamina and luminal border) demonstrated a significant decrease in the proportion of granule-containing (granulated) secretory cells and a corresponding increase in nongranulated secretory cells. By 3 days after HNE injection, the differential count had returned to control levels and cell surface alterations were less evident. By 8 days, the proportion of granulated secretory cells had significantly increased, while that of nongranulated secretory cells had decreased. Many Clara cells developed the characteristics of mucous cells so that mucous cells constituted 57% of the secretory cells compared to 14% for the saline controls. The mucous cells contained an increased number of mucous granules including bizarre forms never seen in controls. By day 16, the average mucous cell proportion had increased to 75%; the mucous cells were larger and contained many more secretory granules than at day 8. At no time was there evidence of overt cell injury or alteration of extracellular connective tissue due to HNE. Basal and pseudobasal cells, distinguished by the presence or absence of hemidesmosomes, did not change as a percentage of total nucleated epithelial cells. Saline had no effect on the differential cell count compared to untreated values. Our results indicate a strong likelihood that HNE causes early discharge of secretory granules and alters the phenotypic expression of Clara cells so that they produce abundant, often abnormal mucous granules. The mechanism of HNE-induced disturbance of epithelial homeostasis is unknown, but the early irregularity of nonciliated epithelial cell surfaces may signify an important event in the evolution of the resultant lesion.

Studies on fine structure and location of lipids in quick-freeze replicas of atherosclerotic aorta of WHHL rabbits.

The fine structure of intracellular and extracellular lipids in the atherosclerotic aorta of Watanabe-heritable hyperlipidemic (WHHL) rabbits was demonstrated by a quick-freeze etching technique. Many lipid droplets, with and without a membrane, were observed in the foam cells. Membrane-free droplets were observed as onion-like structure with a concentric lamellar structure surrounded by 10 nm filaments. Droplets surrounded by a limited membrane probably correspond to lipid-laden lysosomes. In the extracellular connective tissue space, marked accumulation of lipids with a vesicular structure was seen among collagen fibers. The appearance of these lipids was similar to that of lipids in lysosomes of foam cells.

Scar formation after skeletal muscle injury

The formation of a connective tissue scar was studied autoradiographically 2-42 days following standard contusion injury in the gastrocnemius muscle of 30 rats. The rats were injected intraperitoneally with 3H-labeled proline 4 h before the muscle samples were taken. On day 2 the majority of cells in the injured area were identified as inflammatory without radioactive labeling. Abundant labeling was observed, however, over the extracellular substance, and moderate labeling with 3H-proline in the cells identified as fibroblasts or their precursors. During the first 2 weeks the number of fibroblasts increased and the majority of them were labeled with silver grains, which were also present over the connective tissue matrix. Six weeks after injury only a few fibroblasts showed labeling, and no labeling was observed extracellularly in the connective tissue. Thus, it seems that the synthesis of extracellular connective tissue components in injured skeletal muscle occurs as early as 2 days after trauma, is intensive between days 5 and 21 after trauma, and decreases markedly during the following 3 weeks.

Effects of papaverine on smooth muscle cell morphology and vein graft preparation.

Venous injury following mechanical distension and its late sequelae were studied in a canine model. Jugular vein segments distended without and with papaverine (60 mg/100 ml) were compared with nondistended vein segments after they had been placed into the arterial circulation for 1 to 12 months. Arteriography showed no significant statistical discrepancy in luminal diameter of the three vein segments. Complete reendothelialization had occurred in all vein grafts at the time of harvesting. Similar histocytologic features existed in the nondistended segments and in the portion distended with human plasma protein fraction (Plasmanate) plus papaverine (150 to 300 mm Hg). The intimal-medial hyperplasia was characterized by an increase in fibrocellular layers with an elaboration of vasa vasorum. Conversely, collagen and extracellular matrix replaced the myocyte, producing medial fibrosis in vein mechanically distended with Plasmanate alone. Papaverine prevented mechanical stimulation of smooth muscle cells to overproduce extracellular connective tissue elements. Mechanical stretching of intimal and medial myocytes induced fibrogenesis and fibroplasia. Papaverine is useful in vein preparation; it protects the endothelium and smooth muscle cells in the intima and media and prevents leukocyte infiltration and medial fibrosis.

Effects of papaverine on smooth muscle cell morphology and vein graft preparation

Venous injury following mechanical distension and its late sequelae were studied in a canine model. Jugular vein segments distended without and with papaverine (60 mg/100 ml) were compared with nondistended vein segments after they had been placed into the arterial circulation for 1 to 12 months. Arteriography showed no significant statistical discrepancy in luminal diameter of the three vein segments. Complete reendothelialization had occurred in all vein grafts at the time of harvesting. Similar histocytologic features existed in the nondistended segments and in the portion distended with human plasma protein fraction (Plasmanate) plus papaverine (150 to 300 mm Hg). The intimal-medial hyperplasia was characterized by an increase in fibrocellular layers with an elaboration of vasa vasorum. Conversely, collagen and extracellular matrix replaced the myocyte, producing medial fibrosis in vein mechanically distended with Plasmanate alone. Papaverine prevented mechanical stimulation of smooth muscle cells to overproduce extracellular connective tissue elements. Mechanical stretching of intimal and medial myocytes induced fibrogenesis and fibroplasia. Papaverine is useful in vein preparation; it protects the endothelium and smooth muscle cells in the intima and media and prevents leukocyte infiltration and medial fibrosis.

Normal human endometrium. An ultrastructural survey.

An ultrastructural review of the cyclic changes of endometrial surface and glandular epithelial cells, stromal cells and the stromal microvasculature is presented. Endometrial biopsies were collected from infertile patients with endometriosis or tubal dysfunction. During the proliferative phase, the ergastoplasm and Golgi apparatus of surface and glandular epithelial cells become well-developed, while increasing numbers of mitochondria are seen. In the early secretory phase, the postovulatory triad composed of glycogen accumulation, nuclear channel system and giant mitochondria is described and its functional significance is discussed. Premenstrual cellular involution due to enhanced lysosomal activity such as apoptosis and lysosomal wrapping is detailed. Before ovulation, the endometrial stromal cells are primarily involved in the secretion and remodelling of the extracellular connective tissue fibers and ground substance. After predecidual change, the stromal cells exhibit marked macropinocytotic and phagocytotic properties. We suggest that these proteolytic activities of the stromal cells, together with the presence of blood-derived phagocytes, may account for endometrial clearance, since there are no lymphatic channels in the superficial endometrium. According to this hypothesis, tissue fluid may be drained via the fenestrated (venous) endometrial blood capillaries.

Scanning electron microscopy of nasal arterioles effected by vasoactive agents.

Morphological changes in nasal blood vessels induced by alpha- and beta-adrenergic and cholinergic agonists were studied under a scanning electron microscope after removal of extracellular connective tissue and the basal lamina. Arterioles were constricted and dilatated following topical application of alpha-adrenergic and cholinergic agonists. Distinctive morphological changes were observed on their smooth muscle cells. Smooth muscle cells of constricted arterioles had a rugged surface with numerous fine invaginations. However, in dilatated arterioles the cells had a smooth surface and intercellular spaces were clearly revealed. No remarkable changes were produced in smooth muscle cells by the beta-adrenergic agonist. It is concluded that nasal arterioles are sensitive to alpha-adrenergic and cholinergic stimulation but not to beta-adrenergic stimulation.

Alveolar macrophage modulation of proteolysis by neutrophil elastase in extracellular matrix.

An extracellular connective tissue matrix, made up of components found in the pulmonary alveolar interstitium, was generated in vitro and used as a culture surface and substrate for proteolysis by human alveolar macrophages (AM) and neutrophil elastase (NE). The ability of human AM to modulate NE-mediated degradation of elastin and collagen in the surrounding matrix was studied to gain insights into the inflammatory process that accompanies the pathogenesis of emphysema in humans. Neutrophil elastase that had been internalized by AM showed a diminished but more prolonged time course of matrix proteolysis than did a similar amount of NE added to the matrix in the absence of AM. Collagen and elastin degradation were quantitated by release of hydroxylysine and desmosine, respectively, into the culture medium. Significantly more hydroxylysine and desmosine were released by AM that had internalized NE than by AM or by culture medium alone. When 14 X 10(6) AM were added to the extracellular matrix, followed 2 h later by addition of 2 micrograms of NE, collagen and elastin degradation measured at 24 h were not significantly different from that which occurred when matrix was incubated with NE in the absence of AM. Collagen degradation, determined in the same cultures during the period from 24 to 96 h, was significantly greater when matrix was incubated with both AM and NE. These findings suggest that AM can release previously internalized NE in an enzymatically active form and that AM may enhance collagen degradation in matrix that was also exposed to NE.